The proposed project will generate a detailed, mechanism-oriented survey of the behavioral similarities and differences between T cell subsets during primary and secondary immune responses. This information may lead to improved strategies for clinical immunomodulation, e.g. for vaccinations, tumor therapy and treatment of infectious, inflammatory and autoimmune diseases. Summary: T cell-mediated immune responses require contact-dependent information exchange between T cells and antigen (Ag)-presenting cells (APC). Naive T cells (Tn) are primed by mature dendritic cells (DC) that they encounter in secondary lymphoid organs, such as lymph nodes (LNs). Tn continuously recirculate between the blood and LNs, whereas DC collect Ag in peripheral tissues and then access LNs via afferent lymphatics. For example, during a peripheral viral infection, mature DC present viral Ag to CD8+ Tn in the cortex of LNs draining infected tissue. Ag encounter triggers Tn activation, proliferation and differentiation. After a few days, the dividing cells give rise to a burst of cytotoxic and cytokine-producing effector cells (Teff), which can kill APC. While Teff are generally short-lived, a small subset of activated T cells makes a different fate decision and gives rise to precursors of long-lived memory cells. After the height of the early effector response, the pool of Ag-specific T cells contracts, leaving behind a small population of long-lived memory cells, which respond more vigorously than naive T cells when the Ag returns. Memory cells are heterogeneous with regard to their function and tissue distribution. The best studied subset are the central memory cells (Tcm), which recirculate through LNs similar to Tn, whereas effector memory cells (Tem) migrate to peripheral tissues and only access LNs when they become inflamed. Preliminary work for this project has identified what appears to be a third CD8+ memory cell subset, transitional memory cells (Ttm), which is distinct from both Tcm and Tem, but carries features of both. It is widely held that the career decisions taken by T cells are regulated by the spatio-temporal arrangement of interacting communication molecules on the surface of T cells and DC. However, the physical nature and the kinetics of T cell-APC interactions in LNs are poorly understood. In earlier work for this project, a multiphoton intravital microscopy (MP-IVM) model has been developed to study DC and TCR transgenic CD8 T cells in intact popliteal LN of anesthetized mice. This imaging approach produces 3D time-lapse movies of interacting T cells and APC at subcellular resolution. Its use has led to the discovery that CD8+ Tn are activated by DC in LNs in three distinct phases: during phase 1, T cells undergo short, serial encounters with A DCs. The duration of this phase is inversely correlated with Ag dose; phase 2 features prolonged, stable conjugates during which T cells commence cytokine production; and, after the first day, phase 3 is characterized by return to high motility, short contacts with DC, and rapid proliferation. Based, in part, on these findings, the present proposal will pursue two specific aims: 1.) To investigate how Teff and memory cell differentiation is influenced by the kinetics of naive T cell interactions with DC presenting Ag under precisely controlled conditions. 2.) To compare viral Ag-specific CD8+ Tn, Tcm, Ttm and Tem responses to a viral infection in LNs. PUBLIC HEALTH RELEVANCE: CD8 T cells are a subset of immune cells that have a critical role in the host defense against intracellular pathogens and cancer. In order to exert their protective function these cells must be educated by professional antigen-presenting cells, called dendritic cells, in lymphoid tissues. This project explores, at the single-cell level, the mechanisms and consequences through which this education process allows CD8 T cells in lymph nodes to make critical career decisions that determine the magnitude, character and longevity of T cell-mediated immunity. Results from this work will contribute to the development of improved prophylactic and therapeutic vaccines for infections and cancer and may result in novel therapies for inflammatory and autoimmune diseases.